Biochimica et Biophysica Acta 1764 (2006) 743 – 749
http://www.elsevier.com/locate/bba

Oxidation of Arg-410 promotes the elimination of human serum albumin
Yasunori Iwao a , Makoto Anraku a , Keishi Yamasaki a , Ulrich Kragh-Hansen b , Keiichi Kawai c ,Toru Maruyama a , Masaki Otagiri a,⁎a

Department of Biopharmaceutics, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto 862-0973, Japanb
Department of Medical Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmarkc
School of Health Sciences, Faculty of Medicine, Kanazawa University, Ishikawa 920-0942, Japan
Received 20 September 2005; received in revised form 26 December 2005; accepted 4 January 2006
Available online 30 January 2006

Abstract
The effect of the oxidation of amino acid residues on albumin on its in vivo elimination was investigated using mutants and oxidized HSAs.
The single-residue mutants (H146A, K199A, W214A, R218H, R410A, Y411A) and oxidized HSAs were produced by the recombinant DNA
techniques and incubation with a metal ion-catalyzed oxidation (MCO) system for 12, 24, 48 or 72 h. Pharmacokinetics were evaluated in mice
after labeling with 111In. Structural and functional properties were examined by several spectroscopic techniques. Time-dependent increase incarbonyl group content resulted in increase in the liver clearance of oxidized HSAs. Slight decreases in α-helical content as the result of oxidation
was induced by the increases in accessible hydrophobic areas and the net negative charge on the HSA molecule. No significant change in thepharmacokinetics and structural properties was observed for the W214A, R218H and Y411A mutants, but the properties for the H146A, K199A
and R410A mutants were affected (extent of effect: R410A N K199A N H146A). The liver clearance of these proteins is closely correlated tohydrophobicity (r = 0.929, P b 0.01) and the net charge of the proteins (r = 0.930, P b 0.01). The rate of elimination of HSA is closely related to thehydrophobicity and net charge of the molecule. Further, the R410A mutants had a short half-life and structure similar to oxidized HSA after
oxidation. Therefore, the modification of Arg-410 via oxidative stress may promote the elimination of HSA.
© 2006 Elsevier B.V. All rights reserved.
Keywords: Human serum albumin; Oxidation; Arg-410; Elimination; Liver clearance

1. Introduction
In vivo studies with radioisotope-labeled human serum
albumin (HSA) revealed the plasma half-life to be 19 days inhumans [1]. However, it is well known that certain modifications of HSA shorten its half-life in circulating blood [2,3]. Suchchanges in the rate of elimination of HSA as well as of otherserum proteins seems to depend on the individual elimination
process, but the underlying mechanisms have not been clarified
on a molecular basis.
Oxidative stress is increasingly thought to be a key element insome diseases [4–6]. Witko-Sarsat et al. reported the presence ofAbbreviations: HSA, human serum albumin; rHSA, recombinant HSA;MCO12-HSA, MCO24-HSA, MCO48-HSA and MCO72-HSA, HSA oxidized bymetal ion-catalysis for 12, 24, 48 and 72 h, respectively; bis-ANS, 1,1-bis-4anilino-naphthalene-5,5-sulfonic acid
⁎ Corresponding author. Fax: +81 96 362 7690.
E-mail address: otagirim@gpo.kumamoto-u.ac.jp (M. Otagiri).
1570-9639/$ - see front matter © 2006 Elsevier B.V. All rights reserved.
doi:10.1016/j.bbapap.2006.01.011

elevated levels of oxidized protein products, termed advanced
oxidation protein products (AOPP) such as oxidized albumin, inthe plasma of hemodialysis (HD) patients [7]. It has been well
documented that HSA is quite vulnerable to reactive oxygen
species (ROS) [8]. In plasma, all amino acids in a protein aresusceptible to oxidative modification by oxidants such ashydroxyl radicals and hypochlorous acid. Among them, aminoacids, such as cysteine, histidine, lysine and arginine, are more
vulnerable to oxidation than the others [9]. We recently foundthat the modification of these residues on HSA result inconformational changes in cases of uremia [10]. However, theissue of whether the modification of a specific amino acidresidue affects the rate of elimination of HSA is not clear. Sincechanges in hydrophobicity and net charge on a protein surface
affect the in vivo clearance of proteins [11,12], the consequencesof HSA modification, such as oxidation and glycation, may
increase its elimination rate. Sheffield et al. recently reported thatthe half-life of rabbit albumin was reduced to 2.87 days for the744

Y. Iwao et al. / Biochimica et Biophysica Acta 1764 (2006) 743–749

D494N variant [13]. This suggests that the modification of a
specific amino acid residue of HSA could result in an increase in
its rate of elimination.
In the present work, we examined the effect of oxidation on
the plasma half-life and organ uptake of HSA in mice using
mutants and oxidized HSAs. A metal ion-catalyzed oxidation(MCO) system was used for the oxidation, because ferrous ionsin vivo are capable of generating radicals, which can oxidize
proteins such as HSA [14]. On the other hand, in order toinvestigate the amino acids residues related to its elimination,
we prepared six single-residue mutants of HSA, which are
known to be elements of the three important binding sites,
namely His-146 in subdomain IB, Lys-199, Trp-214 and Arg218 in Site-I and Arg-410 and Tyr-411 in Site-II (rHSA) by sitedirected mutagenesis. The structural and functional propertiesof the molecules were also examined by spectroscopic andelectrophoretic analysis.
2. Materials and methods
2.1. Materials and animals
HSA was donated by the Chemo-Sera-Therapeutic Research Institute(Kumamoto, Japan). Fluoresceinamine (isomer II) and the fluorescence probe
1,1-bis-4-anilino-naphthalene-5,5-sulfonic acid (bis-ANS) were obtained from
Sigma (St. Louis, MO, USA). Potassium warfarin (Eisai Co., Tokyo, Japan) andketoprofen (Sanwakagaku Co., Tokyo, Japan) were obtained as pure substancesfrom the manufacturers. 111InCl3 (74 MBq/mL in 0.02 N HCl) was a gift from
Nihon Medi-Physics (Takarazuka, Japan). All other chemicals were also of thehighest grade commercially available, and all solutions were prepared indeionized and distilled water.
Male ddY mice (24–26 g) were purchased from the Shizuoka Agricultural
Cooperative Association for Laboratory Animals (Shizuoka, Japan). Animals
were maintained under conventional housing conditions. All animal experiments were conducted in accordance with the principles and procedures outlinedin the National Institute of Health Guide for the Care and Use of Laboratory
Animals.

2.2. Synthesis of single-residue mutants
The recombinant DNA techniques used to produce wild-type rHSA andthe single-residue mutants H146A, K199A, W214A, R218H, R410A andY411A were essentially the same as described by Watanabe et al. [15]. The
chimeric plasmid (pJDB-ADH-L10-HSA-A) having cDNA for the matureform of HSA along with an L10 leader sequence was a gift from Tonen Co.
(Tokyo, Japan). The mutagenic primers used (underlined letters indicate
mismatches) were 5′-GCCAGAAGAGCTCCTTACTTTTATGCCC-3′ for
H146A, 5′-AAACAGAGACTCGCCTGTGCCAGTCTCC-3′ for K199A, 5′GAGCTTTCAAAGCAGCTGCAGTAGCTCGCCTG-3′for W214A, 5′-GGGCAGTAGCTCATCTGAGCCAGAG-3′ for R218H, 5′-CTATTAGTTTGCTACACCAAG-3′ for R410A, 5′-CTATTAGTTCGTGCCACCAAG-3′ for
Y411A.
The L10-HSA coding region was amplified by PCR with a forward and areverse primer carrying a 5′-terminal EcoRI site and cloned into the EcoRIdigested pKF19k vector (Takara Shuzo Co., Kyoto, Japan). Mutagenesis was
performed with a site-directed mutagenesis kit (oligonucleotide-directed dual
amber method), obtained from Takara Shuzo Co. The mutation was confirmed
by DNA sequencing of the entire HSA coding region with the dideoxy chain
termination method on a PerkinElmer ABI Prism 310 Genetic Analyzer. For
constructing the HSA expression vector pHIL-D2-HSA, a L10-HSA coding
region without or with the desired mutation site was incorporated into themethanol inducible pHIL-D2 vector (Invitrogen Co., San Diego, CA, U.S.A.).
The resulting vector was introduced into the yeast species P. pastoris (strain
GS115) for rHSA expression. The secreted rHSA was isolated from the growth

medium by a combination of precipitation with 60% (w/v) (NH4)2SO4 andpurification on a Blue Sepharose CL-6B column (Amersham Pharmacia Co.,
Uppsala, Sweden). HSA, rHSA and the mutants were defatted using the charcoal
procedure described by Chen [16], deionized, freeze-dried and then stored at−20 °C until used. Density analysis of protein bands stained with Coomassie
Brilliant Blue showed the purity of the recombinant albumins to be in excess of
97%. The molecular mass of all the albumins was assumed to be 67 kDa.

2.3. Synthesis of oxidized HSAs
To prepare MCO-HSA, HSA (300 μM) was incubated in 67 mM sodium
phosphate buffer (pH 7.4) at 37 °C in an oxygen-saturated solution containing
sodium ascorbate (100 mM) and FeCl2 (10 μM) [17]. Ascorbate was added toreduce the Fe3+ formed in the oxidation back to Fe2+. Aliquots were withdrawn
after different time intervals (12, 24, 48, 72 h), and the oxidative process was
terminated by cooling and removing the oxidants by extensive dialysis against
water. The MCO-HSAs and HSA were stored at −20°C until used.

2.4. Carbonyl content determination
Protein carbonyl content was quantified using the method of Climent et al.
[18]. The groups were derivatized with fluoresceinamine and their modified
levels were calculated from the absorbance of the complexes at 490 nm (Jasco
Ubest-35 UV/VIS spectrophotometer).

2.5. In vivo experiments
All proteins were radiolabeled with 111In using the bifunctional chelating
regent DTPA anhydride according to the method of Hnatowich et al. [19], which
has been described in a previous paper [20]. In previous works, we found no
significant changes in pharmacokinetics properties among these albumins, when111
In-labeled mouse, rat, bovine and human serum albumin was administered tomice (unpublished). Therefore, we chose the mouse as a reasonable model forthe study of the pharmacokinetics of HSAs. Mice received tail vein injections of
111
In-labeled proteins in saline, at a dose of 1 mg/kg and were housed inmetabolic cages to allow the collection of urine samples. Urine samples werecollected throughout the 120 min of the experimental period. In the early period
after injection, the efflux of 111In radioactivity from organs is assumed to be
negligible, because the degradation products of 111In-labeled ligands using
DTPA anhydride cannot easily pass through biological membranes [21]. This
assumption was supported by the fact that no 111 In was detectable in the urine
throughout the 120 min. At appropriate intervals after the injection, blood wascollected from the vena cava under ether anesthesia and plasma was obtained bycentrifugation. The liver, kidney, spleen, lung, heart and muscle were excised,rinsed with saline and weighed. The radioactivity of each sample was measured
in a well-type NaI scintillation counter (ARC-500, Aloka, Tokyo).
Pharmacokinetic analyses were performed as follows. The plasma 111In
radioactivity concentrations (Cp) were normalized with respect to the percentageof the dose per mL and analyzed using the nonlinear least-square program
MULTI [22]. The two-compartment model was fitted according to the Akaike
information criterion by Eq (1).
Cp ¼ Aeat þ Bebt

ð1Þ

The half-lives of these HSAs were calculated by the β-phase. The tissue
distribution patterns were evaluated using tissue uptake clearances (CLtissue)
according to the integration plot analysis. CLtissue was calculated using Eq (2).
CLtissue ¼

AUC0t =Ct:
Xt =Ctð2ÞWhere Xt is the tissue accumulation at time t, AUC 0–t is the area under theplasma concentration time-curve from time 0 to t, and Ct is the plasma
concentration at time t. CLtissue was obtained from the slope of the plot of Xt /Ctversus AUC0–t/Ct Mukai et al. [23] previously reported that 111In is not suitablefor evaluating the dynamic phase of a protein for which the in vivo half-life islong. Therefore, we estimated the plasma half-life and liver uptake clearance
within a 120-min period.

Y. Iwao et al. / Biochimica et Biophysica Acta 1764 (2006) 743–749

745

2.6. Effective protein hydrophobicity
The effective hydrophobicity of all the albumins (1 μM), in 67 mM sodium
phosphate buffer (pH 7.4), was estimated using the fluorescent characteristics of
bis-ANS (10 μM) at 25 °C. The compound was excited at 394 nm, andfluorescence spectra were recorded on a Jasco FP-770 fluorescence spectrometer(Tokyo, Japan).

2.7. Changes in protein net charge
Changes in the net charge of albumin, as a result of oxidation or mutation,
were determined by a modification of the capillary electrophoresis method
described by Pande et al. [24]. One mL of a HSA sample (2 μM) was run in 100
mM borate buffer (pH 8.5 and 20 °C), and the migration time determined byusing a CE990/990-10 type capillary electrophoresis from Jasco Co. (Tokyo,
Japan) equipped with a 30-cm capillary having an aperture window of 100 × 200
μm.

Fig. 1. Carbonyl content of MCO-HSAs as a function of incubation time andserum carbonyl content of HD patients (N = 20, from Ref. [10]). The bars
represent standard deviations (n = 4). *P b 0.05 and **P b 0.01 as compared toHSA.

2.8. Ligand binding experiments
Binding of warfarin (5 μM) and ketoprofen (5 μM) to HSA and oxidized
HSAs (10 μM) in 67 mM sodium phosphate buffer (pH 7.4 and 25 °C) was
studied by ultrafiltration. The unbound ligand fractions were separated using theAmicon MPS-1 micropartition system with YMT ultrafiltration membranes by
centrifugation (2000×g, 40 min). The adsorption of warfarin or ketoprofen to thefiltration membranes and apparatus was found to be negligible. The
concentration of unbound ligand was determined by HPLC. The HPLC system
consisted of a Hitachi 655A-11 pump and a Hitachi F1000 variable fluorescencemonitor or a Hitachi 655A variable wavelength UV monitor. LiChrosorb RP-18(Cica Merck, Tokyo, Japan) was used as the stationary phase. The mobile phaseconsisted of 200 mM sodium acetate buffer (pH 4.5)/acetonitrile (40:60, v/v) for
warfarin and of 200 mM sodium acetate buffer (pH 4.5)/acetonitrile (60:40, v/v)
for ketoprofen. The flow rates in both cases were 1 mL/min. Warfarin wasquantitated fluorometrically by using 300 nm and 400 nm for excitation andemission, respectively, and ketoprofen was detected at 257 nm by means of UV
monitoring. The unbound fraction (%) was calculated as follows:
Unbound fractionð%Þ ¼ ½ligand concentration in filtered fraction=total ligand concentrationðbefore ultrafiltrationÞ 100:

2.9. Statistics
Statistical analyses were performed using the Student t-test. A probability
value of P b 0.05 was considered to indicate statistical significance.

3. Results
3.1. Extent of carbonyl content on oxidized HSAs
The oxidation of a protein results in an increase in carbonyl
content. This increase is due to the oxidation of Lys, Arg, or Pro
residues. As seen in Fig. 1, the carbonyl content (nmol/mg
protein) increased significantly with the time of incubation,compared with the level in a control sample of HSA. Data for the
carbonyl content were as follows: MCO72-HSA (5.72 ± 0.23) N
MCO 48 -HSA (5.25 ± 0.16) N MCO 24 -HSA (4.92 ± 0.32) N
MCO12-HSA (3.94 ± 0.31) N HSA (2.36 ± 0.24). These datasuggest that the state of MCO-HSAs accurately reflects theoxidative state of serum proteins in uremia (3.12 ± 1.11 nmol/mg
protein, Fig. 1). Interestingly, the results of amino acid analysis
showed that an exposure to MCO for 48 h incubation induced asignificant change in content of arginine among the basic aminoacids (Arg; MCO48-HSA, 21.0 ± 0.2, HSA, 23.9 ± 0.4 (P b 0.05),
Lys; MCO48-HSA, 57.3 ± 0.5, HSA, 58.0 ± 0.1, His; MCO48HSA, 14.4 ± 0.4, HSA, 15.1 ± 0.4, n = 4).
3.2. Pharmacokinetic properties of oxidized HSAs and mutated
rHSAs
The time courses for radioactivity in mouse plasma after the
intravenous administration of 111In-labeled preparations ofalbumin are shown in Fig. 2. Table 1 shows the half-lives ofthese HSAs calculated by β-phase using the nonlinear leastsquare program MULTI (see Materials and methods). It can beseen from Fig. 2A that the more HSA is oxidized, the faster theradioactively labeled protein disappears from the circulation.
Among the mutations (Fig. 2B), R410A had the mostpronounced effect. Uptake by heart, lungs, spleen and kidneys
were not significantly affected, either by the oxidation or
mutations (data not shown). However, the rapid disappearanceof oxidized HSA from the plasma is accompanied by a very
pronounced increment in liver clearance. It is also apparent thatthe increment depends on the degree of oxidation. The
mutations also showed an increased liver clearance. In
particular, in the case of R410A, liver clearance was increasedby more than 27-fold (Table 1).
3.3. Effect of oxidation and mutation on the net charge ofalbuminAccording to the literature, the formation of MCO-HSA
involves the modification of basic amino acid residues [14,25].
To test whether the oxidation procedure here affected basic
amino acid residues, the net charge of the albumin preparations
was investigated by determining their migration times incapillary electrophoresis. As seen in Fig. 3A, basic amino acidresidues were clearly modified because an increase inmigration time reflects an increase in the net negative chargeof HSA. To identify specific amino acid residues that are
oxidized in producing MCO-HSA, we modified some of theamino acids in HSA by site-directed mutagenesis. From Fig.

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Fig. 2. Relative plasma concentrations of 111In-labeled MCO-HSAs (A) and111
In-labeled rHSA forms (B) after intravenous administration in mice. 111Inalbumin was injected as a bolus through the tail vein, and relative radioactivities
are plotted against time after injection. The bars represent standard deviations(n = 5–6).

3A, it can be seen, that the mutation of H146A and K199A
situated in subdomain IB and the Site-I binding, domain led to
an increased migration time (13.23 ± 0.18 min and 13.83 ± 0.26
min). In contrast, W214A and R218H had no significant effecton the migration of albumin. Of the mutations affecting site II
Table 1
Half-life and liver clearances of oxidized and mutated rHSAs labeled with 111In
in mice
ProteinHalf-life (min)Liver clearance (μL/h)

HSA
MCO12-HSA
MCO24-HSA
MCO48-HSA
MCO72-HSA
Wild-type rHSA
H146A
K199A
W214A
R218H
R410A
Y411A

273 ± 6.25
211 ± 3.31 **187 ± 8.05 **170 ± 8.34 **150 ± 5.09 **272 ± 9.85
238 ± 6.22 *221 ± 5.78 **206 ± 5.26 **274 ± 9.29 *162 ± 9.26 **265 ± 5.75 *13.37 ± 2.57
136.0 ± 3.72 **222.1 ± 2.02 **498.3 ± 8.02 **534.7 ± 4.13 **15.73 ± 5.57
98.31 ± 5.75 *
144.4 ± 6.01 **126.7 ± 6.51 **36.92 ± 8.56 *426.2 ± 8.12 **65.13 ± 8.07 *The data are average values of five or six experiments (± S.D.).
*P b 0.05.
**
P b 0.01 as compared with HSA (oxidized HSAs) or with wild-type rHSA
(mutated rHSAs).

Fig. 3. Electrophoretograms of MCO-HSAs and single-residue mutants (A). The
bars represent standard deviations (n = 4). *P b 0.05 as compared to HSA.
Relationships between protein net charge and liver clearance (B).

binding, the mutation Y411A had no effect, whereas R410A(13.89 ± 0.29 min) had a significant effect on migration.
Furthermore, a change in the net charge on albumin is closely
correlated with that of liver clearance (Fig. 3B; r = 0.930,
P b 0.01). These results suggest that the modification of basic
amino acid residue greatly affects the elimination rate of HSA.
3.4. Effect of oxidation and mutation on the hydrophobicity ofalbumin
The effects of oxidation and mutation on the exposure ofhydrophobic areas were examined using the fluorescence probebis-ANS. The spectra indicate that increased oxidation results ina parallel increase in accessible hydrophobic areas (Fig. 4A). In
contrast, the mutations had a small effect, because only themutations K199A and R410A mutants had an influence on bisANS fluorescence. We also found that the change in thehydrophobic areas on albumin is closely correlated with that ofliver clearance (Fig. 4B; r = 0.929, P b 0.01). These results
suggest that a change in hydrophobic areas greatly affects theelimination rate of HSA.

Y. Iwao et al. / Biochimica et Biophysica Acta 1764 (2006) 743–749

Fig. 4. Effect of oxidation and mutation on the fluorescence of albumin-boundbis-ANS (A). The spectra are averages of three determinations. Relationships
between protein hydrophobicity and liver clearance (B).

3.5. Binding properties of oxidized HSAs
The unique ligand binding properties of albumin can, to agreat extent, be explained by the presence of Site I and Site II
located in subdomain IIA and IIIA, respectively [26]. The
potential effect of oxidation on these sites was examined byusing warfarin and ketoprofen as representative ligands. As seenfrom Table 2, high-affinity binding of warfarin, which takes
place at Site I, was not significantly affected even after
oxidation of HSA for 72 h. In contrast, high-affinity binding of
the Site II-ligand ketoprofen to the MCO-HSAs was greatly
diminished. The diminished binding was already observed foralbumin, which had only been oxidized for 12 h, and the effectincreased further with incubation time (Table 2).

747hydrophobic regions were more exposed and had a negative
charge [10]. However, the issue of whether the modification of a
specific amino acid residue is responsible for this is unclear. In
the current studies, we investigated whether the oxidation of theamino acid residues on albumin affects its elimination in vivo
via the use of mutants and oxidized HSAs.
A significant increase in the carbonyl content of MCO-HSAs
was found with the time of incubation. Furthermore, thecarbonyl content of MCO-HSAs was similar to those of serum
proteins with uremia (Fig. 1). Thus, this result suggests that thestate of MCO-HSAs in vitro reflects the uremic state of serum
proteins in vivo. The rate of protein clearance from the blood
was proportional to the time of oxidation (Fig. 2A). We also
found an increased liver clearance of all of the oxidized
albumins (Table 1). One reason for the shorter half-life andincreased liver clearance of oxidized HSA could be due to
conformational changes induced by changes in the net charge ofthe protein or its hydrophobicity. Another reason could be dueto the modification of one or more essential amino acidresidues. In this connection, it is noteworthy that Segal et al.
[28] reported a correlation between protein hydrophobicity andthe in vivo degradation rates of soluble rat liver proteins. In thepresent work, the oxidation also results in a slight decrease in αhelix content of the protein (MCO72-HSA (58.6 ± 2.9%) b
MCO48-HSA (60.3 ± 2.35%) b MCO24-HSA (62.1 ± 3.21%) b
MCO12-HSA (65.3 ± 2.15%) b HSA (68.3 ± 1.18%). The minor
conformational changes are accompanied by increases in theaccessible hydrophobic areas of the protein (Fig. 4A). As
evidenced by capillary electrophoretic measurements, oxidation
also results in an increase in the negative net charge of HSA(Fig. 3A). Furthermore, taking the results for the oxidized
albumins and the mutants into account, there is a good linear
relationship between liver clearance and protein hydrophobicity(r = 0.929, P b 0.01, Fig. 4B) as well as between liver clearance
and net charge of the protein molecules (r = 0.930, P b 0.01, Fig.
3B). Of the mutations that affect structural properties, only amutation at K199A and R410A had a significant effect on thehydrophobicity and net charge of HSA. According to X-ray
crystallographic analyses those residues are situated inside
cavities or pockets in hydrophobic surroundings and are not in
contact with the solvent [27,29]. Therefore, the effects of thesemutations (Figs. 3A, 4A) on structural properties must be due toindirect effects such as changes in protein conformation and/orchanges in electrostatic bonding patterns between residues.

4. Discussion

Table 2
Binding of warfarin and ketoprofen to oxidized HSAs at pH 7.4 and 25 °C

The long half-life of HSA (t1/2 = 19 days) can be modified indisease states and/or age-related processes, most probably via
the covalent modification of the protein. Our previous work
showed that the oxidation of HSA in vitro results in a more
rapid elimination of the protein from the circulation, a process
which appears to be caused by the modification of basic aminoacid residues such as lysine and arginine [3,27]. Furthermore,
we recently found that the oxidation of HSA in vivo purified
from HD patients was conformationally altered, and that its

ProteinHSA
MCO12-HSA
MCO24-HSA
MCO48-HSA
MCO72-HSA

Free fraction (%)
Warfarin

Ketoprofen

24.32 ± 1.57
28.15 ± 1.87
27.65 ± 2.34
28.23 ± 3.02
27.42 ± 2.15

3.91 ± 0.47
31.4 ± 2.29 *37.8 ± 3.71 *42.3 ± 2.28 **47.8 ± 2.94 **The data are average values of four experiments (± S.D.).
*P b 0.01.
**
P b 0.001 as compared with HSA.

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Thus, the increase in elimination rate may be due to not only tothe modification of basic amino acid residues such as lysine
and/or arginine but also to the modification of a specific site on
HSA. The HSA molecule also contains a free cysteine residue
and six methionine residues. We previously reported that theseamino acid residues were oxidized in the early stage of theoxidation, but that this had no effect on the pharmacokinetics of
HSA [3]. These results strongly support our view that theoxidation of lysine and/or arginine residues affected thepharmacokinetics of HSA, leading to rapid elimination.
In an attempt to determine which lysine and/or arginine
residues were oxidized, we examined high-affinity binding toSite I and Site II on oxidized HSA. High-affinity binding at Site
I was not altered significantly but Site II was significantly
reduced, in the case of oxidized HSA (Table 2). Principally, thesame observation was made when examining Site II binding foralbumins in which Arg-410 and/or Tyr-411 [30] were mutated.
It should be noted that the binding to R410A (Free fraction for
ketoporofen binding; 35.85 ± 4.03%, Ref. [30]) is similar to
those for MCO12-MCO48-HSA (Table 2). Furthermore, aminoacids analysis demonstrates that MCO results in the selective
modification of Arg. At present, it is not possible to conclude
for sure, whether the effect at Site II is caused by
conformational changes at the site, a direct oxidative effecton Arg-410 or a combination of the two. In any event, theeffect of mutating Arg-410 to alanine was dramatic. Ahmed etal. recently suggested that Arg-410 on HSA is modified bymethylglyoxal in vivo [31]. Above findings suggest that Arg410 on HSA may be a residue that is sensitive to physiologicalmodification such as oxidation and glycation. Although thiseffect could be brought about by indirect means, such aschanges in protein hydrophobicity and charge, it is tempting tosuggest that such a pronounced effect is a specific one,
implying that the integrity of Arg-410 is important for both theligand binding properties and the plasma half-life of HSA.
Previous studies have shown that modified proteins, whichcontain oxidized albumin and advanced glycation end products(AGE) albumin, accumulate in liver extremely rapidly after
intravenous administration, due to receptor-mediated endocytosis by nonparenchymal cells, particularly liver endothelial
cells (LECs) [32,33]. Thus, we conclude that oxidized HSA
prepared with the MCO system in this paper may also undergo
scavenger receptor-mediated endocytosis by LECs. Several
scavenger receptors that bind to modified proteins on LECs
have been reported. These receptors include the following: SRA, a scavenger receptor class A for oxidized LDL [34,35];
CD36 and SR-B1, scavenger receptor class B for AGE [36,37];
SREC, a scavenger receptor class F for modified LDL [38];FEEL-1 and FEEL-2, endocytic receptors for AGE [39,40]; andgp18 and gp30, scavenger receptor for chemically modified ofoxidized albumin [41,42]. On the other hand, Matsumoto et al.
reported that SR-A is not involved in the endocytosis of AGE
by LECs using SR-A knock-out mice [33]. Furthermore,
Nakajou et al. reported that CD36 is not involved in the uptakeof AGE by LECs using an anti-CD36 antibody [43]. Therefore,
the molecular nature of the receptors involved in endocytic
uptake of modified proteins is not well understood. In the

future, the receptor responsible for the liver uptake of oxidized
HSA needs to be identified.
In conclusion, the present study has clearly shown that theoxidative modification of a specific site on HSA may lead toconformational changes as well as the rate of elimination of
HSA. In particular, the integrity of Arg-410 is important for theplasma half-life of HSA.
Acknowledgements
This work was supported, in part, by Grants-in-Aid for
Scientific Research from the Ministry of Education, Science,Sports and Culture of Japan (14370759) and by Fonden af 1870.
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